Harpagoside-enriched extract from harpagophytum procumbens and processes for producing same

ABSTRACT

The disclosure relates to extracts from Harpagophytum procumbens with a high harpagoside content, to processes for producing them, such extracts containing no components capable of stimulating the synthesis of thromboxane B2 and cysteinylleucotrienes, and to pharmaceutical compositions containing such extracts.

This application is a 371 of PCT/DE97/00591 filed Mar. 21, 1997.

Due to many years of experience, tea preparations or extracts,respectively, from roots of devil's claw (radix harpagophyti) have beenemployed in the case of dyspeptic conditions as well as in the treatmentof rheumatoid diseases (Sticher, O., DAZ 117 (1977), 1279-1284). Thedrug is composed of the underground parts and mainly of the secondarystorage roots of Harpagophytum procumbens (Volk, O.H., DAZ 104 (1964),573-576; F.C. Czygan, Zeitschrift fur Phytotherapie 8 (1987), 17-20).

M. Caprasse, J. Pharm. BeIg., 1980, 35, 2, 143-149, reviewsHarpagophytum procumbens and its properties. The finely pulverized rootwas extracted with methanol, the resulting extract was evaporated, theresidue added with water, and the resulting aqueous solution wasextracted 3 times with a 4:1 mixture of methylene chloride andn-butanol.

R.E. Moati, FR-A-2 614 791, describes a composition of 400 mg ofHarpagophytum procumbens, 20 mg of selenium and 25 mg of zinc for thetreatment of rheumatism and inflammations.

The commission E of the former Bundesgesundheitsamt took the reports andclinical observations into account by publishing a positive monography“Radix harpagophyti” in 1989. According to this publication,preparations from devil's claw roots are employed in the case ofdyspeptic conditions (daily dose of 1.5 g of the drug) and in thesupportive therapy of degenerative diseases of the motoric system (dailydose of 4.5 g of the drug) (Bundesanzeiger No. 43 of 02/03/1989). Todate it has not been possible to attribute the efficacy to particularingredients.

As the essential ingredients of the extracts there have been mentionediridoid glycosides, in particular harpagoside, and also harpagide andprocumbide. Furthermore, they contain high amounts of sugar (50-60%),fats, waxes, and sterines (Steinegger, Hänsel, Lehrbuch derPharmakognosie und Phytopharmazie, Springer Verlag Berlin, Heidelberg,New York 1988, pp 608-610). Particularly, tea preparations (R.Jaspersen-Schib, DAZ 130 (1990), 71-73; F.-C. Czygan in M. Wichtl,Teedrogen WVA Stuttgart 1989, pp 495-497) as well as capsule and tabletpreparations for oral administration containing simple aqueous oraqueous alcoholic extracts (Chrubasik et al., Forsch. Komplementarmed.1996:3:57-63) are commercially available.

Recently, an antirheumatic effectivity has been shown in a clinicaldouble-blind study. For this purpose, extracts have been employedensuring a dosage of 50 mg of harpagoside per day (S. Chrubasik, R.Ziegler in Phytopharmaka 2—Forschung und klinische Anwendung—Loew,Rietbrock, eds., Steinkopf Verlag Danrstadt 1996 (pp 101-114)).

The conventional pharmaceutical preparations containing simple extractsshow harpagoside contents of 1 to 3% (Chrubasik et al., Forsch.Komplementärmed. 1996:3:6-11) so that as high amounts of extract as 1500to 4500 mg would be necessary to ensure an administration of 50 mgharpagoside/day. This again would require large drug formulations or afrequent or repeated intake of the preparation thereby leading to adecrease in patient compliance.

The object of the present invention is to provide novel extractsenriched in harpagoside content in which ingredients without or withundesired pharmacological effects have been depleted. It is a furtherobject of the invention to provide methods for the preparation of suchextracts as well as drug formulations containing those extracts.

The inyention is based on the surprising finding that aqueous or aqueousalcoholic primary extracts generally having a harpagoside content ofonly 1 to 3% may be strongly enriched in harpagoside according to theinvention by stirring with neat aliphatic alcohol or aliphatic keton ormixtures thereof up to values of at least 5% while ingredients having astimulatory effect on the synthesis of thromboxane B₂ andcysteinyl-leucotrienes are reduced or eliminated.

The term “aqueous alcoholic primary extracts” refers to extractsobtained by extracting radix harpagophyti with a mixture of water andethanol. The aliphatic alcohols contain from 1 to 4 C atoms, thealiphatic ketons contain from 3 to 4 C atoms. Preferably, for stirringthere are used methanol, ethanol, propanol, isopropanol, n-butanol,isobutanol, tert butanol, aceton, butanon and the mixtures thereof.Ethanol is especially preferred. The stirring is performed at atemperature in the range of about 5 to 25° C. The term “stirring” refersto intimate mixing by stirring.

In a preferred embodiment the ground drug is extracted with 20%ethanol/water. Subsequently a concentration step is carried out, and theresulting primary extract is stirred with ethanol 96% at roomtemperature. The soluble fraction is separated from the insolublefraction and dried. It contains at least 5% of harpagoside.

In a preferred embodiment the ground drug is extracted with 20%ethanol/water. Subsequently a concentration step is carried out, and theresulting primary extract is stirred with ethanol 96% at roomtemperature. The soluble fraction is separated from the insolublefraction and dried. It contains at least 5% of harpagoside.

A further surprising finding was that extracts prepared according to themethod described have a higher pharmacological activity in comparison toprimary extracts but also to pure harpagoside. Conventional primaryextracts have been found to contain ingredients leading to a potentstimulation of the synthesis of pro-inflammatory lipid mediators andthereby counteracting the desired antiphlogistic effects. Theseingredients are depleted in the extracts of the invention. The depletionor removal, respectively, of these undesired ingredients is performed bythe stirring according to the invention of the primary extract with thealcohol or keton or the mixtures thereof.

The extracts according to the invention may be used to prepare orallyapplicable solid or liquid drug preparations having an antirheumatic andantiphlogistic activity by adding conventional pharmaceutical adjuvantsin a known manner.

The invention will be further illustrated with respect to the followingExamples:

EXAMPLE 1

30 kg of dry secondary storage roots of Harpagophytum procumbens crushedby grinding were added with 300 kg of completely demineralized waterheated to 85° C. and stirred for two hours. The extract was filteredunder pressure via a plate filter. The plant material was againextracted with 120 kg of hot water at 85° C. for two hours, and theextract was filtered. The extracts from the two extraction stages werecombined: 348 kg. Afterwards, the obtained extract was concentrated in arotary evaporator under vacuum to 19 kg with a dry residue of 60.5%. Theresulting thick extract was dried at a max. of 60° C. and a pressure of20 mbar. This step gave 11.4 kg of dry extract having a harpagosidecontent of 2.25% (as determined by HPLC).

EXAMPLE 2

90 kg of dry secondary storage roots of flarpagopliytum procumbens wereextracted in three batches with hot water according to Example 1. Thecombined extract solutions (1032 kg) were concentrated in a rotaryevaporator under vacuum at 180-220 mbar and 50° C. to 67 kg with a dryresidue of 51.2%. This concentrate, i.e. the primary extract, was addeddropwise under stirring to 350 kg of ethanol 95% by weight at 20° C.(room temperature). For deposition of the precipitate formed the mixturewas left for two hours without agitation. The supernatant was withdrawnand concentrated in a rotary evaporator to a dry residue of 54.4%. Thisconcentrate was dried in a vacuum drying chamber at a maximum of 60° C.and a pressure of 20 mbar affording 7.56 kg of a harpagoside-rich dryextract having a harpagoside content of 7.3% (as determined by HPLC).

EXAMPLE 3

101.5 kg of dry secondary storage roots of Harpagophytum procumbenscrushed via a 7 mm sieve were heated to 75° C. with 1420 kg of ethanol20% by weight for two hours under stirring. After cooling to 50° C., theresidue was filtered and again extracted with 1010 kg of ethanol 20% byweight at 75° C. The combined extract solutions were concentrated in arotary evaporator under vacuum at 180-220 mbar/50° C. to 87 kg with adry residue of 68%. This concentrate (primary extract) was added understirring to 240 kg of ethanol 95% by weight. After the precipitate haddeposited the supernatant was separated and concentrated in a rotaryevaporator under vacuum at 250 mbar to 16 kg. This concentrate was driedin a vacuum drying chamber at a maximum of 65° C. and a pressure of 20mbar affording 8.5 kg of a harpagoside-rich extract containing 12.7% ofharpagoside (as determined by HPLC).

EXAMPLE 4

The precipitate obtained by stirring with ethanol according to Example 3was dried in a vacuum drying chamber at 65° C. and a pressure of 20 mbaraffording 21 kg of an extract fraction containing 0.35% harpagoside.

EXAMPLE 5

1.5 kg of ground dried secondary storage roots of Harpagophytumprocumbens were extracted with 15 kg of ethanol 80% by weight for onehour at 60° C. After cooling, the extract solution was withdrawn via afilter layer. The extract residue was re-extracted with 15 kg of ethanol80% by weight for one hour at 60° C. The combined extract solutions wereconcentrated at the rotary evaporator with a bath temperature of 50-60°C. under a vacuum of 120 mbar until no more ethanol distilled over. Theconcentrate was diluted to a dry residue of 10% by addition ofcompletely demineralized water. This solution (primary extract) wasintimately agitated three times each with 1800 g of n-butanol at roomtemperature. In each case the butanol phase formed (upper phase) wasseparated. At the rotary evaporator the combined butanol phases wereconcentrated to dryness under vacuum at 20-30 mbar. The dry extract wasground and redried in a vacuum drying chamber at 60° C. and 10 mbaraffording 61 g of harpagoside-rich extract having a harpagoside contentof 19.3% (as determined by HPLC).

EXAMPLE 6 Coated Tablet Containiiig the Extract According to theInvention

Coated tablets containing 200 or 400 mg, respectively, of the dryextract of the invention have the following composition:

Devil's claw dry extract according to Example 5 200 mg 400 mg Highlydispersed silicon dioxide 5 mg 10 mg Polyvinyl pyrrolidone (mol. wt. of25,000) 3 mg 6 mg Crosslinked polyvinyl pyrrolidone 5 mg 10 mg Magnesiumstearate 2 mg 4 mg Microcristalline cellulose 35 mg 70 mg

The components were mixed and compressed to form tablets. The tabletswere coated by a film coating on the basis of methyl hydroxypropylcellulose.

Pharmacological Activity of Various Extracts

The pharmacological effects on the formation of pro-inflammatory lipidmediators were examined in whole human blood after stimulation with thecalcium ionophor A23187 (10 mM, 60 min at 37° C. ) according to themethod of I. Weide, K. Tschorn, T. Simmet (Thrombosis Research 67, pp123-134 (1992)). In this model for example glucocorticoids show aninhibitory action. The concentrations of cysteinyl-leucotrienes (LTC₄,LTD₄, and LTE₄) and of thromboxane B₂ were determined in plasma using aradioimmuno assay. Harpagoside or Harpagophytum extracts, respectively,proved to inhibit the biosynthesis of these substances inconcentration-dependent manner. The results are summarized in Table I.It is clear from Table I that the inhibitory effect of the extractsaccording to the invention (prepared according to Example 2 or 3) oncysteinyl-LTs was more pronounced than that of the primary extract(prepared according to Example 1) or of pure harpagoside.

An examination of the precipitate which was removed from the primaryextract according to Example 3 in order to enrich effective ingredients(Example 4) revealed that it has a clear stimulatory effect on thebiosynthesis of cysteinyl-leucotrienes and TXB₂ (Table II) and thereforecounteracts the desired anti-inflammatory effects.

TABLE I Inhibition of the biosynthesis of cysteinyl-leucotrienes andTXB₂ in whole human blood after pre-incubation with harpagoside orHarpagophytum extracts (15 min at 37° C.) and subsequent stimulationwith A23187 (10 mM, 37° C. for 60 min). The half-maximal inhibitoryconcentration (IC₅₀) based on the harpagoside content is shown. IC₅₀TXB₂ IC₅₀ cysteinyl-LTs Harpagoside 48.6 mM   39 mM Harpagophytumextract (primary extract >100 mM 61.7 mM prepared according toExample 1) Harpagoside-rich extract according to 55.3 mM  9.2 mM theinvention (prepared according to Example 2)

TABLE II Stimulation of the biosynthesis of cysteinyl-leucotrienes andTXB₂ in whole human blood after pre-treatment with the extract fractionobtained according to Example 4 (15 min at 37° C.) and subsequentstimulation with A23187 (10 mM, 37° C. for 60 min). The percentage ofstimulation in comparison to a control sample at a concentration of 1mg/ml is shown. IC₅₀ IC₅₀ TXB₂ cysteinyl-LTs Harpagophytum extractfraction of +28.7% +70.5% Example 4

The methods for analysis of thromboxane B₂ and cysteinyl-leucotrienesare detailed in the following references:

Simmet Th., Luck W. (1989), Clotting of whole human blood inducescysteinyl-leucotriene formation. Thromb Res 54:423-433.

Simmet Th., Weide I. (1991), Thromboxane and cysteinyl-leucotrieneformation are differentially activated in spontaneously clotting wholehuman blood in vitro. Thromb Res 62:249-261.

Weide I., Tschorn K., Simmet Th. (1992), EfTects of cyclooxygenaseinhibitors on ex vivo cysteinyl-leucotriene production by whole humanblood allowed to clot spontaneously. Comparison to stimulated blood.Thromb Res 67:123-134.

Weide I., Simmet Th. (1993), Leucotriene formation by peripheralmonocytes in contact-activated human blood, Thromb Res 71:185-192.

Weide I., Romisch J., Simmet Th. (1994), Contact activation triggersstimulation of the monocyte 5-lipoxygenase pathway via plasmin. Blood83:1941-1951.

What is claimed is:
 1. A drug extract from Harpagophytum procumbensobtainable by the steps of: a) extracting the drug from Harpagophytumprocumbens with water or a mixture of ethanol and water, b)concentrating the resulting extract; c) stirring the concentrateobtained at temperatures of 5 to 25° C. with a neat aliphatic alcoholhaving 1-4 carbon atoms or an aliphatic ketone having 3 to 4 carbonatoms or the mixtures thereof; and d) separating the insoluble fractionfrom the obtained soluble fraction, wherein the soluble fraction is thedrug extract and has a strongly reduced content in ingredients havingstimulatory effects on the synthesis of thromboxane B₂ andcysteinyl-leucotrienes.
 2. The extract according to claim 1 which isobtained by the additional step of drying the resulting solublefraction.
 3. The extract according to claim 2 having a content of atleast 5% harpagoside.
 4. A process for the preparation of the drugextract from Harpagophytum procumbens according to claim 1, comprising:a) extracting the drug with water or a mixture of ethanol and water; b)concentrating the resulting extract; c) stirring the concentrateobtained at temperatures of 5 to 25° C. with a neat aliphatic alcoholhaving 1-4 carbon atoms or an aliphatic ketone having 3 to 4 carbonatoms or the mixtures thereof; and d) separating the insoluble fractionfrom the obtained soluble fraction, wherein the soluble fraction is thedrug extract and has a strongly reduced content in ingredients havingstimulatory effects on the synthesis of thromboxane B₂ andcysteinyl-leucotrienes.
 5. The process according to claim 4 wherein forstirring there is used methanol, 96% ethanol, propanol, isopropanol,butanol, butanone, acetone, or a mixture thereof.
 6. A process accordingto claim 4 further comprising drying the resulting soluble fraction. 7.A pharmaceutical composition containing an extract according to any ofclaims 1 to
 3. 8. A method of treating a human suffering from rhematoidarthritis, the method comprising administering an effectiveantirheumactic amount of a compound according to any one of claims 1-3to a patient in need thereof.
 9. A method of treating a human sufferingfrom inflammation and/or fever, the method comprising administering aneffective antiphlogistic amount of a compound according to any oneclaims 1-3 to a patient in need thereof.